The present invention relates to the field of molecular genetics and, in particular, to the production of recombinant Haemophilus influenzae adhesin (Hia) proteins.
Haemophilus influenzae is the cause of several serious human diseases, such as meningitis, epiglottitis, septicemia and otitis media. There are six serotypes of H. influenzae, designated a to f, that are identified by their capsular polysaccharide. H. influenzae type b (Hib) was a major cause of bacterial meningitis until the introduction of several Hib conjugate vaccines in the 1980""s (ref. 1. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Vaccines based upon H. influenzae type b capsular polysaccharide conjugated to diphtheria toxoid (ref. 2), tetanus toxoid (ref. 3 and U.S. Pat. No. 4,496,538), or Neisseria meningitidis outer membrane protein (ref. 4) have been effective in reducing H. influenzae type b-induced meningitis. The other serotypes of H. influenzae are associated with invasive disease at low frequencies, although there appears to be an increase in the incidence in disease caused by these strains as the incidence of Hib disease declines (ref. 5; ref. 6). Non-encapsulated or non-typeable H. influenzae (NTHi) are also responsible for a wide range of human diseases including otitis media, epiglottitis, pneumonia, and tracheobronchitis. The incidence of NTHi-induced disease has not been affected by the introduction of the Hib vaccines (ref. 7).
Otitis media is the most common illness of early childhood, with 60 to 70% of all children, of less than 2 years of age, experiencing between one and three ear infections (ref. 8). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children. H. influenzae infections account for about 30% of the cases of acute otitis media and about 60% of chronic otitis media. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. Furthermore, many of the causative organisms of otitis media are becoming resistant to antibiotic treatment. An effective prophylactic vaccine against otitis media is thus desirable.
During natural infection by NTHi, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and, therefore, potential vaccine candidates. A family of high molecular weight proteins (HMW1 and HMW2) that are important in attachment of NTHi to epithelial cells has been identified in about 70 to 75% of NTHi strains (ref. 9; ref. 10). These high molecular weight adhesins have been shown to afford some protection in the chinchilla model of otitis media (ref. 11). A second family of high molecular weight adhesion proteins has been identified in about 25% of NTHi and in encapsulated H. influenzae strains (ref. 12; ref. 13, ref. 14). The NTHi member of this second family is termed Haemophilus influenzae adhesin or Hia and the homologous protein found in encapsulated strains is termed Haemophilus influenzae surface fibril protein or Hsf. The hia gene was originally cloned from an expression library using convalescent sera from an otitis media patient, which indicates that it is an important immunogen during disease. The prototype Hia and Hsf proteins demonstrate about 82% sequence similarity, although the Hsf protein is considerably larger. The proteins are comprised of conserved amino and carboxy termini and several repeat motifs, with Hsf containing more repeat sequences than Hia. A high molecular weight protein (200 kDa) has also been identified from Moraxella catarrhalis that has some sequence homology with the Hsf and Hia proteins (U.S. Pat. No. 5,808,024).
Since Hia or Hsf is conserved amongst encapsulated strains of Haemophilus influenzae and about 20 to 25% of non-encapsulated strains, and has been demonstrated to be an adhesin, the protein has utility in diagnosis of and vaccination against disease caused by H. influenzae or other bacterial pathogens that produce Hia or a protein capable of raising antibodies specifically reactive with Hia.
A disadvantage of Hia for use as an antigen in diagnosis, for the generation of anti-Hia antibodies useful in diagnosis and as an immunogen in vaccination is the low recovery of the native protein from Haemophilus influenzae species.
It would be advantageous to provide recombinant Hia protein for use as antigens, in immunogenic preparations including vaccines, carriers for other immunogens and in the generation of diagnostic reagents.
The present invention is directed towards the provision of recombinant H. influenzae adhesin (rHia) proteins.
In connection with the provision of such recombinant proteins, the present invention provides certain isolated and purified nucleic acid molecules. Accordingly, in one aspect thereof the present invention provides an isolated and purified nucleic acid molecule encoding a Haemophilus influenzae adhesin (Hia) protein of a strain of Haemophilus influenzae having: (a) a DNA sequence selected from the group consisting of those shown in FIGS. 18, 19, 20, 21, 22, 23, 24 and 25 (SEQ ID Nos: 23, 25, 27, 29, 31, 33, 35, 37); or (b) a DNA sequence encoding a Haemophilus influenzae adhesin (Hia) protein having an amino acid sequence selected from the group consisting of those shown in FIGS. 18, 19, 20, 21, 22, 23, 24 and 25 (SEQ ID Nos: 24, 26, 28, 30, 32, 34, 36, 38).
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode the Hia protein from strain 11 or 33 of non-typeable Haemophilus.
In another aspect of the present invention, there is provided an isolated and purified nucleic acid molecule encoding an N-truncated Haemophilus influenzae adhesin (Hia) protein of a strain of Haemophilus influenzae which is amplifiable by a pair of nucleotides which are selected from the group consisting of SEQ ID No: 7 and SEQ ID No: 15; SEQ ID No: 9 and SEQ ID No: 15; SEQ ID No: 11 and SEQ ID No: 15; SEQ ID No: 13 and SEQ ID No: 15.
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode an N-truncated Hia protein from strain 11 or 33 of non-typeable Haemophilus, starting at codon V38.
The plasmid vector incorporating the isolated and purified nucleic acid provided in accordance with these aspects of the invention may have the identifying characteristics of a plasmid which is selected from the group consisting of:
DS-2008-2-3 as shown in FIG. 1A
DS-2186-1-1 as shown in FIG. 5A
DS-2201-1 as shown in FIG. 5A
DS-2186-2-1 as shown in FIG. 5A
DS-2168-2-6 as shown in FIG. 5A
The vector provided herein may include the cer gene from E. coli. Accordingly, in another aspect of the present invention, there is provided a vector for transforming a host, comprising a nucleic acid molecule encoding a full-length or N-truncated Haemophilus influenzae adhesin (Hia) protein, a promoter for expression of said full-length or truncated Hia protein and, optionally, the cer gene of E. coli. The vector may be a plasmid vector or other non-replicating vector, which may have the identifying characteristics of a plasmid vector which is selected from the group consisting of:
BK-96-2-11 as shown in FIG. 6A
DS-2242-1 as shown in FIG. 7A
DS-2242-2 as shown in FIG. 7A
DS-2340-2-3 as shown in FIG. 8A
DS-2447-2 as shown in FIG. 9A
DS-2448-17 as shown in FIG. 9B
The vectors provided herein may comprise a replicating vector, including a vector from Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus.
Any of the vectors provided herein may be employed to transform a suitable host cell for expression therein of a protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus, which may be in full-length or truncated form. Such host conveniently may be E. coli. Such expression may be under the control of the T7 promoter and expression of the recombinant Hia from the transformed host may be effected by culturing in an inducing concentration of lactose or other convenient inducing agent.
The present invention further includes, in a further aspect thereof, a recombinant protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable Haemophilus strain producible by the transformed host, particularly E. coli, provided herein. Such Hia protein may be provided in the form of an immunogenic fragment or adhesin-functional analog of the recombinant protein.
The recombinant Hia proteins, full-length or N-truncated, provided herein are useful as antigens in immunogenic composition, carriers for other immunogens, diagnostic agents and in the generation of diagnostic agents. The nucleic acid molecules which encode the Hia protein, full-length or N-truncated, also are useful as probes for diagnostic use and also in immunogenic compositions.
The present invention, in an additional aspect thereof, provides an immunogenic composition, comprising at least one immunologically active component which is selected from the group consisting of an isolated and purified nucleic acid molecule as provided herein and a recombinant protective Hia protein, full-length or N-truncated, of a strain of Haemophilus, as provided herein, and a pharmaceutically-acceptable carrier therefor.
The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host to provide protection against disease caused by H. influenzae. For such purpose, the compositions may be formulated as a microparticle, capsule, ISCOM or liposome preparation. The immunogenic composition may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces.
The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant or at least one cytokine. Suitable adjuvants for use in the present invention include (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein and other adjuvants.
Advantageous combinations of adjuvants are described in copending U.S. patent application Ser. No. 08/261,194 filed Jun. 16, 1994 and Ser. No. 08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (WO 95/34308 published Nov. 21, 1995).
In accordance with another aspect of the invention, there is provided a method for generating an immune response in a host, comprising the step of administering to a susceptible host an effective amount of the immunogenic composition as recited above. The immune response may be humoral or a cell-mediated immune response. Hosts in which protection against disease may be conferred include primates, including humans.
The present invention includes, in a yet additional aspect thereof, a method for the production of a protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus influenzae, which comprises:
transforming a host, such as E. coli, with a vector comprising a nucleic acid molecule encoding an N-truncated form of the Haemophilus influenzae adhesin protein as provided herein,
growing the host to express the encoded truncated Hia, and
isolating and purifying the expressed Hia protein.
The encoded truncated Hia may be expressed in inclusion bodies. The isolation and purification step may be effected by disrupting the grown transformed cells to produce a supernatant and the inclusion bodies containing the Hia, solubilizing the inclusion bodies after separation from the supernatant, to produce a solution of the recombinant Hia, chromatographically purifying the solution of recombinant Hia free from cell debris, and isolating the purified recombinant Hia protein.
The vector transforming the host cell, such as E. coli, may include the T7 promoter and the E. coli or other host cell may be cultured in the presence of an inducing amount of lactose or other convenient inducing agent.
The strain of Haemophilus influenzae herein may be selected from the group of non-typeable strain consisting of strains 11, 33, 32, 29, M4071, K9, K22 and 12. Specific nucleic acid sequences for the gene encoding the Hia protein from such strain are provided herein and are described below.
The nucleic acid molecules provided herein are useful in diagnostic applications. Accordingly, in a further aspect of the invention, there is provided a method of determining the presence, in a sample, of nucleic acid encoding a Haemophilus influenzae adhesin protein, comprising the steps of:
a) contact the sample with a nucleic acid molecule as provided herein to produce duplexes comprising the nucleic acid molecule encoding the Hia protein of a strain of Haemophilus present in the sample and specifically hybridizable therewith; and
b) determining the production of the duplexes.
In addition, the present invention provides a diagnostic kit for determining the presence, in a sample, of nucleic acid encoding a Haemophilus influenzae adhesin protein, comprising:
a) a nucleic acid molecule as provided herein;
b) means for contacting the nucleic acid molecule with the sample to produce duplexes comprising the nucleic acid molecule and any such nucleic acid molecule; and
c) means for determining production of the duplexes.
The recombinantly produced truncated Hia proteins provided herein also are useful in diagnostic: applications. Accordingly, in another aspect of the invention, there is provided a method of determining the presence of antibodies specifically reactive with the Hia protein in a sample, comprising the steps of (a) contacting the sample with the recombinant Hia protein provided herein to provide complexes of the recombinant Hia protein and any such antibodies present in the sample specifically reactive therewith; and (b) determining production of the complexes.
Advantages of the present invention include:
an isolated and purified nucleic acid molecule encoding a Haemophilus influenzae adhesin protein or a fragment or an analog of the Hia protein;
recombinantly-produced Hia proteins, free from any other Haemophilus proteins; and
diagnostic kits and immunological reagents for specific identification of Haemophilus.